apoptosis and oncotic necrosis apoptosis vs oncotic necrosis initiation execution apoptosis...

Apoptosis and Oncotic Necrosis: The final common pathways of liver injury

Hartmut Jaeschke

Department of Pharmacology, Toxicology & Therapeutics

University of Kansas Medical Center Kansas City, KS

Fibrosis: Response to Cell Injury

Rockey & Friedman, 2006

Chronic Cell Injury: Apoptosis and Oncotic Necrosis

• cellular condensation (cell shrinkage)

• membrane blebbing, but no loss of integrity

• aggregation of chromatin at the nuclear membrane

• formation of membrane bound vesicles (apoptotic bodies)

• no disintegration of organelles; organelles remain intact

• swelling of the cell and lysis

• loss of membrane integrity

• flocculation of chromatin

• no vesicle formation, complete lysis

• disintegration (swelling) of cell organelles

Morphology

Apoptosis Oncotic Necrosis

Hepatocellular Apoptosis

H & E

Galactosamine (500 mg/kg; 6 hr)

Hepatocellular Oncotic Necrosis and Inflammation

H & E

Bile Duct Ligation 3 days

Apoptotic Cell Death: Caspases

Proenzyme (zymogen)

prodomain large subunit linker domain small subunit

Peptidase 1 496

DED DED Pro-Caspase 8 Initiator Caspase

Death effector domainDED

Peptidase 1 277 Pro-Caspase 3

Effector Caspase

Cysteine-dependent aspartase domainPeptidase

PeptidaseCARD 1 416 Pro-Caspase 9

Initiator Caspase

Caspase recruitment domainCARD

Activation of Caspases Proenzyme (zymogen)

Active Caspase (tetramer)

prodomain large subunit linker domain small subunit

Activation of Caspases Proenzyme (zymogen)

Active Caspase (tetramer)

prodomain large subunit linker domain small subunit

0 20 40 90 120 Time (min)

p32

p11 Fas-Ab Jo-2

Proenzyme

Active Fragment

0

2 0 0

4 0 0

6 0 0

Activation of Caspases

0 20 40 90 120 Time (min)

p32

p11

(ΔF/min/mg protein)

600

400

200

0

*

*

*

DDDD DD

FAS-L FAS-L

DDDD DD

FA D

D FA

D D

FA D

D FA

D D

Cas 8

FAS-L

DDDD DD

FA D

D FA

D D

FA D

D FA

D D

Cas 10

C as

p1 0

Casp 6

C asp

8Bid

Ba xB ak

C

C

C

C

C

C

C

C C

C

C

C C

C

Casp 7 Casp 3A

pa f1

A pa

f1

C as

p 9

C as

p 9

C

C C

C

CAD

En do

G En

do G

CAD

En do

G

A IF

A IF

D ia

bl o

D iablo

C

B ak B ak

B ax

B ax

Caspase Substrates

CAD

CARD

C

C

CARD

DISC

FAS-L FAS-L

dATP

dATP

Fas-mediated Apoptosis Signaling in Hepatocytes

CAD ICADCAD

Bid

Bcl-2

Ba k

C

C

C C

C

C

C

C

C

C

C

C Casp 3

C

C

C C

EndoG EndoG

EndoG

EndoG

AIF

Diablo

C

Caspase 3 SubstratesAIF

cIAPDiablo

Intrinsic Pathway of Apoptosis

Ca

Ca

Ca

Ca

AIF

Diablo

Diablo C

AIF

C

C

C

C

C

C

C C

CC

C

C

BaxBakBax

Bak

AIF Diablo

Ca Ca

Ca

Ca

Ca Ca

Ca Ca

Ca Ca

PTP

Bcl-XL

Bcl-2 Bax

Bax

Bax

28S

eIF2α

PT P

Cathepsin

C

A pa

f1CARD

Apaf1CA R

D

Apaf1 CA RD

A paf1

CARDCasp 9

Ca sp

9C asp

9

C

C C

Bax BakBax

Bak

CaCa Ca

Ca

Casp 9

C C

m-Calpain

Casp 12

p53Bax

Caspase Targets in Apoptosis Casp10

Casp 6

Casp 8

Casp 7

Casp 3

A pa

f1

A pa

f1

C as

p 9

C as

p 9

CARD C

CARD

CARD Casp 2

MDM2Fodrin

Prese- nelin2 Gelsolin

Actin Lamin A

DNA fragmentation and chromatin condensation

Cytoskeleton Cell shape and membrane

blebbing

CAD

Trans- glutaminase

Topo- isomerase

PARPDNA-PKEndoGICAD

Keratin- 18

Gas2

Cell cycle and other

p21

FAK

hnRNP

β-catenin

NuMASREB1

Calpastatin

EMAP II

Rock-1

DNA Fragmentation

1 2 3 4 5 6 7 8 9 10 11 12 13

MW Controls G/ET

0

200

400

600

800

1000

(% Vmax)

0 0 20 40 90 120

600

400

200

800

1000 *

*

*

Time (min)

DNA LadderAnti-Histone ELISA

CADCAD

DNA Strand Breaks: TUNEL Assay

Terminal deoxynucleotidyl transferase- mediated dUTP nick end labeling

GalactosamineGalactosamine (500 mg/kg; 6 hr)(500 mg/kg; 6 hr)

Characteristic Features of Oncotic Necrosis

• Morphology: cell swelling, cell contents release, karyolysis

Mechanism is dependent on the insult

Oncotic Necrosis: Acetaminophen- induced Hepatotoxicity

Cell Swelling, Karyolysis

Cell Contents Release : Plasma ALT > 3000 U/L)

DNA Strandbreaks during Oncotic Necrosis

TUNEL Assay

CV

4 h APAP 6 h APAP

DNA Strandbreaks: Oncotic Necrosis vs Apoptosis

TUNEL Assay

CV

6 h Gal/ET 6 h APAP

DNA Fragmentation: Apoptosis vs Oncotic Necrosis

1 2 3 4 5 6 7 8 9 10 11 12 13

MW Controls G/ET

DNA LadderAnti-Histone ELISA

APAP

D N

A F

ra gm

en ta

tio n

(% c

on tro

l)

0

500

1000

1500

2000

C G/E G/E ZVAD

AAP AAP ZVAD

AAP GSH

*

* * #

#

1 2 3 4 5 6 7 8 9

0 0.5 1 2 3 4 6 G/E

Acetaminophen (h)

p32

p11

Caspase-3 Processing during Acetaminophen Toxicity

Lawson et al., Toxicol Appl Pharmacol 156: 179-86, 1999 Gujral et al., Toxicol Sci 67: 322-8, 2002

Ba xB ak

C

C

C

C

C

C

C

C C

C

C

C C

C

C

C C

C

EndoG

En do

G En

do G

EndoG

En do

G

EndoG

A IF

A IF

D ia

bl o

D iablo

C

B ak B ak

B ax

B ax

AIF

C

Diablo AIF

EndoG

DNA Fragmentation: Nuclear Translocation of Mitochondrial Intermembrane Proteins

Diablo

C C

Nucleus

Mitochondria

Control

5 mM AAP, 6 h

Endonuclease G

Bajt et al., Toxicol Sci 94: 217-225,2006

Cytochrome c

Bax

Bid tBid

Co ntr

ol AP

AP 2

h

AP AP

4 h

Co ntr

ol AP

AP 2

h AP

AP 4

h

Mitochondria Cytosol

APAP-induced Bax and tBid Translocation to Mitochondria

APAP Hepatotoxicity in Bax Gene Knockout Mice

P la

sm a

A LT

(U /L

)

0

1000

2000

3000

4000

5000

6000

7000

*

WT Bax-/-

Bajt et al., J Pharmacol Exp Therap, in press, 2007

APAP Hepatotoxicity in Bax Gene Knockout Mice

P la

sm a

A LT

(U /L

)

0

1000

2000

3000

4000

5000

6000

7000

*

WT Bax-/-

Bajt et al., J Pharmacol Exp Therap, in press, 2007

WT

Bax-/-

NAPQI APAP GSH ↓

Protein Arylation

P4501.

2.

Bax

Cyt c / Smac AIF Endonuclease G

ONOO-

Nucleus

DNA- Strandbreaks

ATP ↓

Caspase Activation

ATP ↓

PARP Activation

DNA Repair

DNA- Fragmentation

Chromatin Condensation

NAD+ Depletion

ATP ↓ ⇐ MPT

? Mechanism of Mechanism of APAP ToxicityAPAP Toxicity

Toxicol Sci 89: 31-41, 2006

O2 – NO

Bax MPT

Protein Nitration

Apoptosis vs Oncotic Necrosis

Initiation Execution

Apoptosis Apoptosis ATP high